Cone photoreceptors (cones) serve a critical function in human vision that is underscored by the dramatic impairments in sight that occur if these cells die. For instance, retinitis pigmentosa and macular degeneration are two such diseases in which substantial loss of vision correlates with the loss of cones. One promising therapy for these diseases is to replace lost cones with new ones produced inside or outside of the patient. However, the fundamental molecular mechanisms underlying the genesis of vertebrate cones are still unclear. Without this information, a rational strategy to produce cones is unavailable. The long-term goal is to identify the gene regulatory networks that promote cone genesis, and to devise methods based on this knowledge to make new cone cells for cell replacement therapy. The overall objective of this proposal is to functionally dissect the transcriptional networks that allow a specific sub-population of retinal progenitor cells to preferentially generate two rare retinal cell types, cones and horizontal cells. This proposal will test the central hypothesis that the transcription factors Otx2 and Onecut1 are the master regulators of this specific cone/horizontal cell progenitor cell. The rationale for undertaking this study is that by understanding the transcription factor networks that promote the formation of these progenitor cells and ultimately cone photoreceptor cells, methods for generating new cone photoreceptors in vitro or in vivo will become possible. To test this hypothesis, two specific aims are proposed: 1) Identify the transcription factors that establish the cone/horizontal progenitor cell state and promote cone genesis and 2) Define critical parameters of Onecut1 and Otx2 expression that induce cone/horizontal progenitors and differentiated cones from other retinal progenitor cell types. The first aim will determine the genes expressed specifically in cone/horizontal progenitor cells and the extent to which this molecular signature is generated by the Onecut1 and Otx2 transcription factors. In addition, it will identify the transcription factor pathways downstream o Onecut1 that repress rod photoreceptor genesis. The second aim will test the extent to which Onecut1 and Otx2 can reprogram other retinal progenitors into the specific type that generates cones and horizontal cells and ultimately drive the cone differentiation program, while repressing rod genesis. This approach is innovative because it will functionally investigate a specific sub-population of retinal progenitor cells at the whole transcriptome level. In addition, t will dissect and explore the most upstream transcription factor network involved in cone genesis. At the completion of this project, new avenues will be available to understand the biology of specific retinal progenitor types, as well as the early steps in cone and rod photoreceptor development. The research proposed here is significant because it is expected to reveal novel, fundamental insights into the genesis of cone photoreceptors. It is expected that this knowledge will have translational potential for the development of new therapies for human blindness.